Messenger RNAs for the milk proteins, casein and alpha-lactalbumin, were isolated and partially purified from lactating mouse mammary gland. The translation of poly(A)+mRNA in a cell-free system yielded three casein polypeptides and a putative precursor form of alpha-lactalbumin. Analysis of the milk protein mRNAs during the development of mammary gland demonstrated that casein mRNA increased progressively during pregnancy, amounting to 50-60% of the total mRNA activity during lactation, whereas alpha-lactalbumin mRNA remained at low levels during pregnancy and increased sharply during lactatin, amounting to 0.2-0.4% of the total mRNA activity. These results indicate that synthesis of milk protein in lactating tissue are closely parallel with the level of corresponding mRNAs. Addition of vanadate was found to enhance the stimulatory effect of insulin on DNA synthesis in cultured mouse mammary gland. Vanadate, however, inhibited insulin-dependent casein synthesis and blocked the mitogenic action of Li+ on mammary cells. These results are in line with our hypothesis that ions play an important regulatory role in the hormone-dependent development of mammary gland.